A2A Adenosine Receptor Antagonists: Are Triazolotriazine and Purine Scaffolds Interchangeable?

Abstract

The A_2A adenosine receptor (A_2AAR) is one of the four subtypes activated by nucleoside adenosine, and the molecules able to selectively counteract its action are attractive tools for neurodegenerative disorders. In order to find novel A_2AAR ligands, two series of compounds based on purine and triazolotriazine scaffolds were synthesized and tested at ARs. Compound 13 was also tested in an in vitro model of neuroinflammation. Some compounds were found to possess high affinity for A_2AAR, and it was observed that compound 13 exerted anti-inflammatory properties in microglial cells. Molecular modeling studies results were in good agreement with the binding affinity data and underlined that triazolotriazine and purine scaffolds are interchangeable only when 5- and 2-positions of the triazolotriazine moiety (corresponding to the purine 2- and 8-positions) are substituted.

Keywords: A2A adenosine receptor antagonists; anti-Parkinson agents; anti-inflammatory agents; molecular modeling; purine derivatives; triazolotriazine derivatives.

Figure 1

Structures of A_2AAR antagonists.

Figure 2

Structures of the designed compounds: (a) purine derivatives; (b) triazolotriazine derivatives.

Scheme 1

Reagents and conditions: (a) 4-methoxyphenethylamine at 130 °C for 24 h, 86% yield; (b) NBS, dry DMF, r. t. for 30 min, 44% yield; (c) HBr 48% at 100 °C for 2 h, 23, 10, and 32% yields for compounds 9, 10, 11, respectively; (d) 2-tributylstannylfuran, [(Ph)₃]₂PdCl₂, dry THF at reflux for 4 h (12, 35% yield) and 6 h (13, 67% yield).

Scheme 2

Reagents and conditions: (a) hydrazine hydrate, toluene, 0 °C for 1 h, r. t. for 16 h, 83% yield; (b) m-CPBA, DCM, from 0 °C to r. t., 80% yield; (c) 14, 170 °C, 6.5 h, 22% yield; (d) EtOH, NaH, 24 h, r. t., 32 and 5% yields for compounds 18 and 19, respectively; (e) m-CPBA, THF of 2 h, tyramine, Et₃N, 40 °C, 12 h, 10% yield; (f) Et₃SiH, Pd/C 10%, THF, 8 h, r. t., 44% yield.

Figure 3

Ortep style view of compound 17. Ellipsoids are shown at 50% probability level. Selected bond distances (Å) and angles (°): C1-N1 1.323(2), C1-N6 1.353(2), C1-S1 1.7794(16), C2-N6 1.332(2), C2-N5 1.344(2), C2-N2 1.3728(19), C3-N5 1.317(2), C3-N4 1.365(2), C3-S2 1.7480(16), C4-N3 1.311(2), C4-N4 1.325(2), C4-N2 1.3721(19), C5-S2 1.797(2), N1-N2 1.3748(17); N1-C1-N6 118.47(14), N1-C1-S1 118.59(12), N6-C1-S1 122.64(12), N6-C2-N5 129.47(14), N6-C2-N2 109.12(13), N5-C2-N2 121.41(14), N5-C3-N4 128.40(14), N5-C3-S2 120.45(12), N4-C3-S2 111.14(11), N3-C4-N4 123.52(15), N3-C4-N2 119.13(14), N4-C4-N2 117.35(14), C1-N1-N2 99.58(12), C4-N2-C2 121.84(13), C4-N2-N1 127.06(13), C2-N2-N1 110.91(12), C4-N4-C3 117.44(13), C3-N5-C2 113.48(13), C2-N6-C1 101.90(13).

Figure 4

Effect of 13 on cell viability and NO production in BV-2 cells. (A) Cells were treated with 50–5000 nM of 13 for 24 h, and MTT assay was used to measure cell viability. (B) Cells were treated with 10 nM of 13 before activation with 100 ng/mL LPS for 24 h and NO release was measured by the Griess assay. Each bar represents means ± SEM of at least three independent experiments. Data were analyzed by one-way ANOVA followed by Bonferroni’s test. * p < 0.05 vs. CTRL; ° p < 0.05 vs. LPS; § p < 0.05 vs. 10 nM.

Figure 5

Expression of IL-1β, IL-6, COX-2, and iNOS in BV-2 cells treated with 13 and ANR 94. Cells were treated with 10 nM 13 or 10 nM ANR 94 for 24 h, exposed to 100 ng/mL LPS for 24 h and cytokines and enzymes mRNA levels were measured by RT-PCR. Data are expressed as relative abundance compared to untreated cells. Each bar represents the mean ± SEM of three independent experiments. Data were analyzed with one-way ANOVA followed by Bonferroni’s test. * p < 0.05 vs. CTRL; ° p < 0.05 vs. LPS; § p < 0.05 vs. ANR 94 + LPS.

Figure 6

Expression of NLRP3 and IL-10 in BV-2 cells treated with 13. Cells were treated with 10 nM 13, exposed to 100 ng/mL LPS for 24 h and NLRP3 and IL-10 mRNA levels were measured by RT-PCR. Data are expressed as relative abundance compared to untreated cells. Each bar represents the mean ± SEM of three independent experiments. Data were analyzed with one-way ANOVA followed by Bonferroni’s test. * p < 0.05 vs. CTRL; ° p < 0.05 vs. LPS.

Figure 7

General binding mode of the synthesized compounds at the A_2A AR (pdb: 5NM4) binding cavity, with indication of some key receptor residues. (A) Compound 13 is shown in green, with the co-crystallized ligand ZM241385 (purple) for comparison; (B) Docking conformations of compound ANR 94 (5, purple); (C,D) Docking conformations of compound 21 (cyan) with representation of the two observed potential binding modes.

Figure 8

Interaction energies (represented as kcal mol⁻¹) calculated with the aid of the IF-E 6.0 tool at the A_2AAR (A), A₁AR (B), and A₃AR (C) binding sites. Compounds ZM241385 and 13 were submitted to analysis. Receptor residues in proximity to the compound region corresponding to the 9-ethyl substituent of 13 were considered. In the panel at left, there is a representation of the analyzed residues and their position with respect to the 9-ethyl group (surface representation); the labels indicate the A_2AAR residues. See text for details.